Loss of SIRT4 Promotes the Self-Renewal of Breast Cancer Stem Cells
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Theranostics 2020, Vol. 10, Issue 21 9458 Ivyspring International Publisher Theranostics 2020; 10(21): 9458-9476. doi: 10.7150/thno.44688 Research Paper Loss of SIRT4 promotes the self-renewal of Breast Cancer Stem Cells Lutao Du1, Xiaoyan Liu1, Yidan Ren1, Juan Li1, Peilong Li1, Qinlian Jiao1, 2, Peng Meng3, Fang Wang4, Yuli Wang1, Yun-shan Wang1 and Chuanxin Wang1 1. Department of Clinical Laboratory, The Second Hospital, Cheeloo College of Medicine, Shandong University, 247 Beiyuan Street, Jinan, Shandong, 250033, China. 2. International Biotechnology R&D Center, Shandong University School of Ocean, 180 Wenhua Xi Road, Weihai, Shandong 264209, China. 3. The Medical Department of IVD Division, 3D Medicines, Inc., Pujiang Hi‑tech Park, Shanghai 201114, China. 4. Institute of basic medicine, The Second Hospital, Cheeloo College of Medicine, Shandong University, 247 Beiyuan Street, Jinan, Shandong, 250033, China. Corresponding authors: Yun-shan Wang, Department of Clinical Laboratory, The Second Hospital, Cheeloo College of Medicine, Shandong University, 247 Beiyuan Street, Jinan, Shandong, 250033, China. E-mail: [email protected]; Phone Number: 86-531-85875118; Chuanxin Wang, Department of Clinical Laboratory, The Second Hospital, Cheeloo College of Medicine, Shandong University, 247 Beiyuan Street, Jinan, Shandong, 250033, China. E-mail: [email protected]; Phone Number: 86-531-85875118. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2020.02.08; Accepted: 2020.07.07; Published: 2020.07.25 Abstract Rationale: It has been proposed that cancer stem/progenitor cells (or tumor-initiating cells, TICs) account for breast cancer initiation and progression. Sirtuins are nicotinamide adenine dinucleotide (NAD+)-dependent class-III histone deacetylases and mediate various basic biological processes, including metabolic homeostasis. However, interplay and cross-regulation among the sirtuin family are not fully understood. As one of the least studied sirtuin family members, the mitochondrial sirtuin SIRT4 is a tumor suppressor gene in various cancers. However, its role in cancer stemness, as well as initiation and progression of breast cancer, remains unknown. Methods: The expression of SIRT4 in breast cancer was analyzed using the TCGA breast cancer database and 3 GSEA data. Normal breast epithelial cells MCF10A and breast cancer cell lines MCF-7, MDA-MB-231, BT549, MDA-MB-468 were used to establish SIRT4 gene knockdown and corresponding overexpression cells. Identified MTT cytotoxicity assays, cell invasion and motility assay, sorting of SP, confocal immunofluorescence microscopy, mouse mammary stem cell analysis, glutamine and glucose production, clonogenic and sphere-formation assay, mass spectrometric metabolomics analysis and ChIP-seq to further explore SIRT4 biological role in breast cancer. Results: We elucidated a novel role for SIRT4 in the negative regulation of mammary gland development and stemness, which is related to the mammary tumorigenesis. We also uncovered an inverse correlation between SIRT4 and SIRT1. Most importantly, SIRT4 negatively regulates SIRT1 expression via repressing glutamine metabolism. Besides, we identified H4K16ac and BRCA1 as new prime targets of SIRT4 in breast cancer. Conclusions: These results demonstrate that SIRT4 exerts its tumor-suppressive activity via modulating SIRT1 expression in breast cancer and provide a novel cross-talk between mitochondrial and nuclear sirtuins. Key words: SIRT4, SIRT1, breast cancer, cancer stemness, glutamine metabolism Introduction Sirtuins (SIRTs) are nicotinamide adenine stress-responsive proteins that direct various post- dinucleotide (NAD+)-dependent class-III histone translational modifications of histones and down- deacetylases (HDACs) that belong to the silent stream non-histone targets, the seven mammalian information regulator 2 (SIR2) family. As the sirtuins (SIRT1–7) are considered to be master http://www.thno.org Theranostics 2020, Vol. 10, Issue 21 9459 regulators of several fundamental biological This study is to identify a mechanism linking processes, including cell death and survival, decreased SIRT4 expression to breast tumor-initiating chromatin remodeling, energy metabolism, aging, cells (BTICs) regulation and cancer progression. Here, inflammation, and tumor development [1-3]. we show that SIRT4 possesses its tumor-suppressive Among the sirtuin family, SIRT1 is the founding effect on breast cancer by inhibiting glutamine and the most extensively studied member. SIRT1 is metabolism and thereby affecting the protein levels of ubiquitously expressed in the nucleus but shuttles SIRT1, which modulates the acetylation patterns of between the nucleus and cytoplasm [4]. Similar to histones H4 and regulates stemness via BRCA1. other HDACs, aberrant SIRT1 expression is reported in multiple human malignancies, namely, gastric, Materials and Methods colon, prostate, and ovarian cancers, as well as breast Chemicals and Antibodies cancer [5-11]. The implications of SIRT1 in breast cancer occurrence and progression have been Lipofectamine 2000 transfection and TRIZOL LS investigated over recent years. However, the exact reagents were bought from Invitrogen (Grand Island, role is still controversial due to its contradictory NY, USA). The DAB substrate kit has been purchased functional roles. from Vector Laboratories, Inc (Burlingame, CA, USA). Unlike SIRT1-3 and SIRT7, which are primarily Abcam (Cambridge, MA, USA) provided antibodies lysine deacetylases, SIRT4 serves as both an ADP- towards SIRT4 (#: ab10140), SIRT1 (#: ab189494), ribosyl-transferase and lysine deacetylase. It is a H4K16ac (#: ab109463), H3K9ac (#: ab272105), Oct4 relatively unstudied member of the SIRT family [12]. (#: ab19857), SOX2 (#: ab97959). Nanog (#: 8822), SIRT4 is located within mitochondria and regulates BRCA1 (#: 9010) and β-actin (#: 4970) antibodies were catabolism of multiple nutrients [13]. Despite the obtained from Cell Signaling Technology (Danvers, regulatory function in metabolism, SIRT4 was found MA, USA). Unless, in any other case noted, all other to be involved in mediating cell cycle progression and used chemicals were from Sigma (St. Louis, MO, signaling pathways like MEK/ERK or AMPK/ USA). mTORC [14-16]. So far, emerging evidence on the role Cell lines and cell culture of SIRT4 as a tumor suppressor gene has been shown. Normal mammary epithelial cell line MCF10A According to The Cancer Genome Atlas (TCGA) and breast cancer cell lines MCF-7, MDA-MB-231, database analysis, SIRT4 mRNA levels in human BT549, MDA-MB-468 and HEK 293 Phoenix ampho gastric, breast, bladder, colon, thyroid, and ovarian cells were purchased respectively from the Cell Bank cancers were decreased compared with normal tissues of different types culture collections of Shanghai [17, 18]. Decreased SIRT4 protein levels were Institute of Cell Biology and Chinese Academy of associated with poor prognosis in colon, lung, and Sciences. As earlier mentioned, cell lines were esophageal cancer [16, 19, 20]. Intriguingly, although regularly cultured. Typically, cell lines were kept at 37 more tumor tissue samples were found SIRT4 positive °C in an environment comprising 5% CO2 in than adjacent nontumor tissue samples by immuno- Dulbecco's modified Eagle medium or RPMI 1640 histochemical staining, low SIRT4 expression was (Gibco) supplemented with 10% fetal bovine serum. associated with poor overall survival in breast cancers Cells were passed every 1-2 days to keep logarithmic patients, especially in Luminal A patients [21]. growth. All these cell lines were regularly screened However, critical mechanisms linking reduced SIRT4 for mycoplasma contamination by using the expression to breast cancer progression remain Universal mycoplasma detection kit, which was unclear. It has therefore been proposed that loss of obtained from ATCC (Manassas, VA), and the last BRCA1 function in basal stem cells results in tumor mycoplasma test was conducted in May 2017. formation associated with a block in luminal Mycoplasma-free cell lines have been used in all our differentiation [22]. experiments. In contrast, recent work showed an increase in luminal progenitor numbers in the breast tissue of Patients and specimens BRCA1 mutation carriers and a correlation between Randomly, samples were gathered from patients the gene expression profile of normal human luminal with breast cancer who underwent curative resection progenitors and basal-like breast cancers. Compared with informed consent between 2010 and 2015 at Qilu with most sporadic breast cancers, those arising in Hospital, Shandong University. All tissues were carriers of BRCA1 mutations usually have distinctive collected immediately after tumor resection, snap- pathological characteristics. A study suggests that a frozen into liquid nitrogen, and then stored at -80 °C. role for BRCA1 in the determination of stem-cell fate Follow-up information was summarized at the end of may explain this phenomenon [23]. December 2017 with an average observation interval http://www.thno.org Theranostics 2020, Vol. 10, Issue 21 9460 of 75.4 months. Study protocols were endorsed by with a Coherent Enterprise II laser-emitting MLUV. Shandong University's Hospital Ethics Committee. The Hoechst 33342 emission was first divided using a Based on the Helsinki declaration, written informed short-pass